Electrophoresis is a well-established technique for the separation and analysis of mixtures. Electrophoresis involves the migration and separation of molecules in an electric field based on differences in mobility. Many different forms of electrophoresis have been developed to permit the separation of different classes of compounds. These forms include free zone electrophoresis, gel electrophoresis, isoelectric focusing, and isotachophoresis. These techniques can be performed in tubes or channels of micrometer cross-sectional dimensions in what is referred to as capillary electrophoresis (CE). Capillary electrophoresis offers advantages over larger scale systems with regard to assay time and electrophoretic resolution because high electrical field strengths can be used and the technique is readily automatable.
CE is a powerful separation technique and can be used, for example, to separate an antibody-antigen complex from either the unbound form of the antigen or the antibody, thereafter permitting quantitation. When the interaction between analyte and binding partner, e.g., antibody and antigen, is highly specific, the combination of this highly specific reaction with conjugation techniques for attaching a detectable moiety to binding partners makes it possible to use a variety of assay techniques to detect analytes in complex biological samples such as body fluids. For example, U.S. Pat. No. 4,486,530 discloses use of monoclonal antibodies and detectable moieties to determine the presence and/or concentration of IgE in a sample. In general, CE assays have more favorable characteristics such as shorter assay time, less sample volume requirements, low reagent usage, and potentially enhanced sensitivity. See, e.g., Reif et al., Analytical Chemistry 66:4027-4033 (1994).
One of the key factors in successfully employing conventional CE or other electroseparation techniques for immunoassays is the ability to electrophoretically separate unbound and bound forms of antibody. Despite the high resolution power of electroseparation techniques, however, this is not a readily or reliably achievable requisite. A major reason for this is that the antigen, the antibody, or both may exhibit significant heterogeneity due to the presence of variants, isoforms, differences in glycosylation, etc. This means that both the unbound and the complexed form of the antibody migrate non-uniformly, thus producing broad poorly-defined distributions upon electroseparation analysis rather than sharply discernible peaks as desired. Consequently, detection and/or quantitation of unbound versus bound species is difficult and/or unreliable. Thus, for certain analytes, especially large biomolecules, conventional electroseparation methods may not offer significant advantages for clinically useful diagnostic immunoassay applications.
Electroseparation has also been used to analyze nucleic acid analytes as disclosed in WO 93 20236, entitled "Probe Composition and Method." The probes described therein contain nucleic acid analytes and a polynucleotide binding partner attached thereto. The polynucleotide component of the probes is attached to a detectable label as well as a size and/or charge modifier to assist in the electrophoretic fractionation of the individual polynucleotide component upon its release from the probe. Analysis of nucleic acid analytes is accomplished indirectly by correlating the presence/absence of an individual released polynucleotide with the presence/absence of a particular nucleic acid analyte.
Some practitioners have also tailored the electrophoretic mobility of a labeled antibody by attaching charged groups to the same labeled molecule. See, e.g., Chen and Evangelista, Clinical Chemistry 40:1819-1822 (1994); Chen and Sternberg, Electrophoresis 15:13-21 (1994). On the one hand, this results in the mobility of labeled antibody being different from that of the corresponding unlabeled antibody. On the other hand, however, tailoring the charge characteristics of the labeled antibody is not ideal because the ultimate objective of the assay is to separate the two labeled species formed in a typical binding assay of the sort disclosed in the above-mentioned references, i.e., labeled antibody-antigen complex and unbound labeled antibody. According to the methodologies set forth in the above-mentioned references, both such species will be influenced by the charge tailoring, thereby undermining efforts to differentiate between labeled species. Additionally, while the above-described references demonstrate some success using a single antibody having dual modifications, i.e., having both a detectable moiety and a charged moiety attached thereto, the analytes detected are small, low-molecular weight analytes (morphine, PCP, digoxin), with the assay performed in a competitive format, which are typically more conducive to CE analysis than more complex biological macromolecules. See also Evangelista et al., American Clinical Laboratory 14(2):27-28 (1985).